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Thaumarchaeota DS1: Nitrogen metabolism through less energy expenditure

By: Vicky Lopez

Microbiology

Faculty Advisor: Dr. José R. de la Torre

The Thaumarchaeota phylum is one that has been recently recognized as its own in the archaea domain. The analysis of genomic data has facilitated the classification of organisms that have not been cultivated in lab or seen under a microscope. The organism to be studied is Thaumarchaeota Dragon Spring 1 (DS1). It’s genome was obtained from a hot spring in Yellowstone. Using comparative genome analysis little by little there is more being learned from this organism that helps imply what functions it possesses. The focus in this experiment is nitrogen metabolism. DS1 does not reduce nitrogen to ammonium like many of its phylum members. On the other hand, organisms that can fix nitrogen have the advantage of surviving when ammonium is not present. There is high availability of ammonium in hot springs while in the environment of other close relatives of DS1 have other sources available.

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The Metallo-beta-lactamase Enzyme Facilitates in Thaumarchaeota archaeon DS1(Dragon Thaumarchaeon) Antibiotic Resistance

By: Alex Chong

Cellular & Molecular Biology

Faculty Advisor: Dr. José R. de la Torre

Thaumarchaeota archaeon DS1 is a recently discovered archaeon found in the Dragon Spring of Yellowstone National Park. Being a thermoacidophile, DS1 possesses some unique abilities that should be investigated. DS1 may potentially be utilizing Metallo-beta-lactamases (MBL) to confer itself antibiotic resistance. MBL superfamily of proteins all use the MBL fold (a four-layered beta-sandwich fold with two mixed beta-sheets flanked by alpha-helices) as a catalytic unit with either Zinc or Iron ions bound at the active site. It possesses the ability to cleave the beta-lactam ring structure, as well as the structure of one of the last resort antibiotics, carbapenem. Although unlikely that carbapenems exist in hot springs, studying this putative MBL protein in DS1 may provide insights into the evolutionary history and functional mechanism of MBL molecules. I will investigate the possible origin of MBL proteins in DS1 by comparing the putative protein sequences in DS1 to that of bacteria and archaea known to contain such a protein by utilizing pBLAST. I will construct a phylogenetic tree of the putative MBL protein in DS1 and other MBL proteins found in other organisms to elucidate any evolutionary connections.

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Nitric Oxide Involvement in Early Chicken Embryo Myogenesis

By: Noah Kipper, Alex Chong, Anders Peterson, and Fernando R. Curiel

Cellular & Molecular Biology

Faculty Advisor: Dr. Wilfred Denetclaw

During skeletal muscle regeneration, nitric oxide (NO) activates muscle stem cells called satellite cells to become proliferating myoblasts that later differentiate and fuse into skeletal muscle fibers. Skeletal muscle formation begins in the embryo in dorsal dermomyotome layer of somites and are single fibers called myotome. The muscle stem cells in the dermomyotome are regulated by morphogenic signals and growth factors for proliferation and differentiation but myotome development is not coincident with somite formation. Instead, there is a delay of myotome until the embryo has produced 16 somites, and then myotome formation is rapidly made in these existing cranial somites in caudal-ward direction. The reason myotome formation is induced may be due to NO signal as played in satellite cell activation. To investigate, we used somite cell cultures derived from caudal somites, stages I-IV (no myotome are produced), in embryos with 10-24 somites. NO and regulators of its canonical signaling pathway tested the ability of NO to signal muscle formation in cultured somites as revealed by titin immunofluoresce and by muscle fiber morphology. Our results show NO involvement in signaling of cultured somites for myogenesis. Supported IRA Awards.

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Understanding the effects of histone variants on chromatin compaction in Caenorhabditis elegans

By: Israel Saucedo

Cellular & Molecular Biology

Faculty Advisor: Dr. Diana Chu and Dr. Geeta Narlikar (UCSF)

The compaction of an organism’s genome allows for the regulation of gene expression and proper development. To accomplish this, cells incorporate H2A histone variants, which alter inter- and intranucleosomal interactions to influence chromatin dynamics in different cell types. For example HTZ-1, an H2A.Z homolog in C. elegans, localizes to the promoter regions of developmental genes where it may poise genes for expression. Interestingly HTZ-1 has also been shown to suppress the expression of cell cycle genes in C. elegans. On the other hand, HTAS-1, a sperm-specific H2A variant in C. elegans, has been shown to localize to condensing chromatin during spermatogenesis. How H2A variants are able to function in distinct chromatin processes remains elusive. We hypothesize that the variations in amino acid sequence at specific domains of the H2A variants confer structural differences to the proteins. Thus, these domains are responsible for the different functions of H2A variants in chromatin dynamics. To test this, we reconstituted C. elegans nucleosomes in vitro. We show both HTZ-1 and HTAS-1 confer stability to the nucleosome when compared to canonical H2A using fluorescence resonance energy transfer (FRET). Furthermore, our preliminary data shows that the C-terminal domain of HTZ-1 is responsible for the increased nucleosomal stability. While these data provide insight into the complex role of H2A variants, its interpretation is limited to individual nucleosomes. To better understand the global impacts of H2A variant incorporation in C. elegans development, we are assembling arrays of nucleosomes in vitro. This will allow us to study the biophysical characteristics imparted by HTZ-1 and HTAS-1 on the compaction of larger chromatin structures. The degree of compaction of the arrays will be assessed by sedimentation velocity experiments. To address how the domains of H2A variants influence chromatin compaction, we will perform domain-swapping experiments and assess their role in this process. The results from these experiments will show how structural features of H2A histone variants correlate with their influence in chromatin dynamics. This will provide mechanistic insight into the compaction of chromatin, a process crucial for proper gene expression and survival of the organism.

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Measuring Changes of Tissue Depth Markers in an Expressive Face

By: Parham Koohbor

Cellular & Molecular Biology

Faculty Advisor: Gloria Nusse

Analyzing human facial expressions is important for many fields from treatment of facial paralysis to rigging jobs for animators. The underlying mechanisms of facial expression are dependent upon the anatomical features of the head such as the skulls and muscles of the face. There are many other features of the face that can be used to predict an expressive face, such as the use of including tissue depth markers to predict the changes of facial morphology. This project has formulated a method to measure changes at specific tissue depth landmarks, which are anthropometrically meaningful in an expressive face. For this study the measurements were taken on a face reconstructed from real CT scan data. While this project relied on a limited source of data from one face it is the hope of the authors that access to more CT scan data measurements from a large pool of people would have potential to be used as reference values for reconstructing an expressive face.

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Determination of the Membrane Topology of Porcupine Protein

By: Gabriel Fraley

Cellular & Molecular Biology

Faculty Advisor: Dr. Laura Burrus

WNT signaling is critical for proper embryonic development and adult tissue homeostasis. WNT proteins require palmitoylation for optimal secretion and receptor binding. Porcupine (PORCN), a membrane bound O-acyltransferase (MBOAT) family member is the protein that palmitoylates WNT. PORCN is localized to the Endoplasmic Reticulum (ER), Golgi apparatus, and plasma membrane. Though PORCN is known to mediate proper tissue patterning in developing embryos, its topology is poorly understood. Determining the topology of PORCN may have useful applications in areas of embryonic development studies, and development of targeted cancer therapies. Although numerous bioinformatic algorithms that predict membrane topology are available, no clear picture for PORCN has emerged. Therefore, our goal is to test experimentally PORCN to establish a working topological model. Our data using Myc-tagged PORCN orient the N- and C- termini towards the lumenal and cytosolic regions of the ER, respectively. To further determine topology, we are subcloning PORCN constructs with epitopes, transfecting Cos-7 cells and utilizing immunofluorescence to visualize the orientation of FLAG tagged epitopes within the cytosolic or lumenal spaces of the ER, or both. FLAG tags were inserted at amino acid positions 118 and 145 to determine orientation of these protein motifs in the ER. Preliminary data shows the loop being analyzed to be cytosolic, with additional visual and quantified data indicating a possible dual topology.

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Predicted mechanism for domain movement between two conformational states of the Mycobacterium tuberculosis drug target fadD32 enzyme

By: Holland Page

Cellular & Molecular Biology

Faculty Advisor: Dr. Misty Kuhn

Enzymes from the adenylation enzyme superfamily are capable of performing two distinct reactions: (1) activate fatty acids for synthesis of complex lipids using an acyl-adenylate intermediate and (2) transfer of the activated fatty acyl chain to the pantotheine moiety of either Coenzyme A or an acyl carrier protein (ACP). The enzyme transitions between these two reactions by moving its highly flexible C-terminal domain between two different conformations—an adenylation and thioester conformation. The fatty acyl-AMP ligase fadD32 protein from Mycobacterium tuberculosis belongs to this superfamily of enzymes and is important in the cascade of reactions to synthesize mycolic acids for its cell wall. This enzyme has been identified as a drug target for the treatment of tuberculosis; therefore, and understanding of its structure and transition mechanism between different conformations is important for further drug discovery efforts. Since the enzyme has only been crystallized in the adenylation conformation, we used pymol to examine crystal structures of five other enzymes from this superfamily to propose a transition between adenylation and thioester conformations for the fadD32 protein. Specific and distinct interactions between the N- and C-terminal domains occur at each conformation, and we propose that these interactions may be ideal targets for disrupting conformational changes of this protein for future drug design efforts.

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Phylogeography of Blood Parasites in Bird’s of Papua New Guinea

By: Brett K. Morris

Cellular & Molecular Biology

Faculty Advisor: Dr. Ravinder Sehgal

Avian Malaria (Plasmodium) and other blood parasites, such as Haemoproteus, are causing havoc in island populations across the Pacific. These parasites threaten many bird species, including those that are vulnerable and critically endangered. To investigate the diversity and prevalence of these parasites in pristine and understudied island populations, we screened birds from Papua New Guinea for Haemoproteus and Plasmodium using a nested PCR. Positive samples were sequenced and phylogenetic trees were created to determine if geography affected the parasite evolution. For Haemoproteus, prevalence exceeded 18.2% in all birds, with one avian family, Melanocharitidae (Berrypeckers and Longbills) exhibiting 90.9% prevalence. For Plasmodium, the causative agent in Malaria, we found an overall prevalence of 4.1%, with the avian family Meliphagidae (Honeyeaters) having the highest prevalence of 17.4%. Interestingly, the Psittacidae family (Parrots) showed no infections. Statistical analysis of diversity mapped against location for all Haemoproteus species did not reveal geography having a great influence on parasite evolution. However, there was a subtree that showed statistical relevance suggesting that geography influenced its evolution. Taken together, these data reveal evolution patterns of these parasites in birds, allowing for better decisions to be made when setting up conservation sites in the future for preservation of biodiversity hotspots. /

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TMEM16A : chloride channel inhibitors

By: Eric Truong

Biochemistry

Faculty Advisor: Dr. Marc Anderson

My project is focused on TMEM16A (ANO1), a calcium-activated chloride channel (CaCC) expressed in secretory epithelia, smooth muscle, and other tissues. I am synthesizing small-molecule inhibitors, which targets the TMEM16A activators. The synthesized analogues may be useful for drug therapy of cystic fibrosis, dry mouth, and gastrointestinal hypomotility disorders, and for further pharmacological dissection of TMEM16A function.

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Exploring potential kinetic mechanisms for newly identified threonine N-acetyltransferases from Clostridium difficile and Staphylococcus aureus

By: David Tran

Biochemistry

Faculty Advisor: Dr. Misty Kuhn

Gcn5-related N-acetyltransferases (GNATs) are part of an extensive superfamily of enzymes that catalyze the transfer of an acetyl group from acetyl-coenzyme A to an amino group of an array of substrates. GNATs are abundant in all organisms, yet the functions for many of these enzymes remain unknown. In general, GNAT substrates are small molecules like antibiotics and proteins. Protein acetylation can occur on alpha amino groups of N-terminal amino acids or on epsilon amino groups of lysine residues within the protein. Recent studies have shown that N-terminal alpha amino acetylation of proteins is present in prokaryotes; a function previously regarded as present only in eukaryotes. Although prokaryotic proteins are N-terminally acetylated, the enzymes that catalyze this process have not been identified. Our previous substrate screening efforts identified two GNATs from Clostridium difficile and Staphylococcus aureus as being capable of acetylating the amino acid L-threonine, potentially implicating them in N-terminal protein acetylation. To improve the fundamental understanding of how these proteins catalyze their reactions, we kinetically characterized these enzymes. Additionally, we tested different kinetic models using non-linear regression analysis to determine the potential kinetic mechanism(s) for the enzymes. Our results show that a bireactant random steady-state model is most compatible with our data for both enzymes, indicating that bacterial threonine GNAT enzymes use a similar mechanism to catalyze their reactions.

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Designing a Heme Protein without Nitrite Reductase Activity

By: Jessica Bow

Biochemistry

Faculty Advisor: Dr. Raymond Esquerra

The nitrite reductase activity of heme proteins has been shown to play key roles in physiology. Deoxygenated hemoglobin in particular is proposed to support vasodilation during hypoxia by converting nitrite (NO2- ) to nitric oxide (NO). We measured the nitrite reductase activity of several mutants (H64A, H64Q, H64V, L29F, L29Wand H64L) using a spectrophotometric assay. We determined that the rate of nitrite reductase activity depends on the ability of the H64 residue to form a hydrogen bond with the transiently bound nitrite-iron moiety. We additionally demonstrated that the size of the distal pocket also controls reactivity, with larger residues forming a steric barrier hindering the reaction from proceeding. We designed a double mutant, H64L/L29W, which essentially has no nitrite reductase activity. This mutant is potentially useful in measuring NO levels in the presence of nitrite. Understanding how the protein matrix controls the nitrite reductase chemistry of heme proteins is essential in understanding how these proteins generate NO physiologically. NO is an important signaling molecule included in various signaling pathways, and thus, with furthered understanding therapeutics can be designed based on the nitrite reductase activity of heme proteins.

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Impact of Hydrogen Sulfide on the Enzymatic Activity of Neuronal Nitric Oxide Synthase

By: Lara Manimbao

Biochemistry

Faculty Advisor: Dr. Raymond Esquerra

Nitric oxide (NO) and hydrogen sulfide (H2S) both have a role as biological signaling molecules in mammals, functioning similarly as muscle relaxants in the vascular system through different mechanisms. NO is synthesized from L-arginine by different isoforms of nitric oxide synthases (NOS) while H2S is synthesized from cysteine by cystathione beta synthases. Although there is a clear correlation in physiological response from both these gasostranmitters, there is much debate on the mechanism of their interactions. The goal of this project is to determine if H2S can directly impact the activity of neuronal nitric oxide synthase (nNOS). Our working hypothesis is that hydrogen sulfide will inhibit nNOS activity by modifying reactive thiols and a zinc cofactor in the nNOS homodimer. We measure nNOS activity as a function of added H2S. The rate of NO production from nNOS is followed through an assay converting oxyhemoglobin to methemoglobin which is observed at 401 nm by UV/VIS spectrophotometry.

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Towards Establishing the Mechanism of Pyridine Nucleotide Recycling in the Styrene Catabolic and Detoxification Pathway

By: Madison Huynh, Leslie Galvez, and Kyle Kulinski

Biochemistry

Faculty Advisor: Dr. George Gassner

Despite its carcinogenic effects, styrene has been widely used in major reinforced-plastic and styrene-butadiene rubber industries. This reliance has a direct effect upon human health as styrene exposure degrades the body by causing DNA adducts and genetic damage in lymphocytes. To mitigate these impending effects, a focus is being placed on the Pseudomonas putida S12 styrene degradation process in biotechnological applications. The catabolic and detoxifying characteristics of styrene monooxygenase (SMO) enable it to epoxidize styrene to phenylacetic acid (PAA) and create central metabolites. The styrene metabolic pathway recycles NADH/NAD+ via a coenzyme exchange reaction. In the first step of the pathway, the flavin reductase component of SMO oxidizes NADH as a source of electrons for the styrene epoxidation reaction. NAD+ generated in this reaction is transferred to the terminal enzyme in the styrene catabolic pathway, phenylacetaldehyde dehydrogenase (PADH), where it is used to oxidize phenylacetaldehyde (PAL) to phenylacetic acid (PAA). This study investigates the structure of potential pyridine nucleotide-exchange complexes formed in recycling of NADH/NAD+ in metabolism. To analyze this interaction, PDB files of existing structures were submitted to the ClusPro2 structural bioinformatics server to establish possible SMOB-PADH interaction interfaces. The models returned by ClusPro2 were sorted based on the distances separating the NAD+/NADH binding pockets and in this way models most representative of the pyridine nucleotide exchange complex were identified. To test the validity of the computationally theorized SMOB-PADH interaction interface, we plan to use steady state fluorescence quenching measurements as a means of detecting this protein-protein interaction. This will be done by labeling the active site of PADH with TEMPO maleimide, a cysteine-reactive reagent that houses an unpaired electron. The unpaired spin in the active site of PADH will quench the intrinsic fluorescence of proximal NADH and FAD bound to SMOB. The measurement of fluorescence quenching in this way will allow the interaction of the enzymes to be established experimentally. In the present work, we demonstrate the viability of the labeling strategy by using UV/Vis-stopped-flow apparatus to establish conditions for labeling the active site thiol during enzyme turnover.

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Role of Flavins in Stabilizing and Nucleating the Folding of Styrene Monooxygenase Reductase

By: Natalia Achtar-Zadeh and Angelina Motiee

Biochemistry

Faculty Advisor: Dr. George Gassner

Styrene is a synthetic molecule with importance in the manufacturing of plastic, rubber and resins. Although styrene has proven to be useful, it has also been identified as a potential environmental pollutant and carcinogen. Due to the associated dangers of styrene exposure, studies of its degradation pathway are essential. Our study involves characterizing the unfolding and refolding kinetics of the enzymatic protein, styrene monooxygenase (SMO), which is capable of safely degrading styrene. SMO is a two-component flavoprotein that has subcomponents SMOA epoxidase and SMOB reductase, which work to catalyze the flavin and NADH specific epoxidation reaction of styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway. Our studies investigate N-terminally Histidine-tagged version of SMOB reductase (NSMOB) and its flavin coenzyme specificity of the folding reaction, as well as optimal protocol conditions. Comparing flavins FAD, FMN, and Riboflavin, we find FAD to be the most effective folding-catalyst for NSMOB. The catalytic activity of NSMOB was studied as a function of urea concentration and in this way it was established that in the presence of FAD, NSMOB folding transitions occur at midpoint urea concentration of 3M. Urea concentration-jump studies by rapidly mixing NSMOB with urea solutions containing FAD and NADH on the stopped flow spectrophotometer monitoring the kinetics of NADH oxidation at 340 nm show that kinetics of the reversible folding of NSMOB is complete in the first 200 ms after mixing. A refolding rate constant of 16.7 turnover/sec and an unfolding rate constant of 36.2 turnovers/second were deduced. In addition, we find that washing frozen inclusion-body pellets containing NSMOB with cold buffer and centrifugation before unfolding with 8M urea dramatically increased kinetic activity relative to an unwashed pellet. This suggests a soluble matter in the unwashed pellet that inhibits activity and can be remedied by our washing protocol. /

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Rapid Method to Determine Iron in Vitamins and Supplements Using a Portable X-Ray Fluorescence Analyzer

By: Swee K. Chew

Biochemistry

Faculty Advisor: Dr. Pete Palmer

The goal of this project was to develop and apply a new X-Ray Fluorescence (XRF) method to rapidly quantify iron in vitamins and supplements. Samples were prepared by grinding to a fine powder and diluting into cellulose to reduce matrix effects. Standards in the range of 0-2000 ppm iron were prepared by mixing and grinding known masses of iron oxide and cellulose. Samples and standards were placed into sample cups and analyzed using a portable XRF analyzer. Calibration curves showed excellent linearity and demonstrated the viability of the method used to prepare standards. Analysis of a NIST Standard Reference Material (SRM) demonstrated the accuracy of this method and gave relative error of less than 5%. Precision of the method was good, with RSDs around 5% or less for samples and standards. Application of this method to determine iron levels in several different vitamin and iron supplement products showed some interesting results. Iron levels varied by as much as 30% from pill to pill, and in such cases accurate quantification requires a sample preparation procedure requiring the grinding of 10 or more pills to obtain a representative sample for analysis. A men’s vitamin product labeled to contain no iron was found to contain ~200 ppm iron. This method is deemed to be superior to conventional atomic spectrometry techniques in that it does not require sample digestion, is simpler, cheaper, and involves measurement times on the order of one minute per sample. Ultimately, this method may find use in a regulatory setting to rapidly assess the levels of iron, calcium, or even potentially toxic elements in drugs and supplements.